This invention generally relates to the diagnosis of plant diseases; and, more specifically, to a method especially well suited for the field diagnosis of plant diseases, and to a kit including the materials needed to carry out that method.
Plant diseases cause a tremendous amount of damage and economic loss each year. For example, it certain types of grass diseases, such as pythium blight, become wide spread over a particular area, it may be necessary to remove and to replace all the grass in the affected area. Even if this is not necessary, an extensive amount of time and treatment may be required to control the disease.
The magnitude of the problem is caused, in part, by the fact that heretofore it has been very difficult or prohibitively expensive to diagnose most plant diseases until the symptoms of the diseases became visible. In particular, very few plant diseases can be diagnosed in a laboratory before the symptoms of the diseases become visible in the field, and most such laboratory tests are expensive and time consuming. Consequently, these tests are not normally performed until there is visible evidence in the field that a plant may be infected; and as a result, in most instances, by the time a disease is finally diagnosed, it is quite widespread.
Numerous comparatively simple and inexpensive diagnostic tests have been successfully developed using antibodies to detect human and animal diseases in very early stages of infection. To do this, a first antibody, which is reactive with a selected antigen that is, or is associated with, a disease causing organism, may be immobilized on a solid support member; and then a sample liquid solution, which is suspected of harboring the selected antigen, is contacted with the immobilized antibody. If the selected antigen is present in the sample liquid solution, the antigen reacts with and becomes bound to the first antibody to form an antigen-antibody binary complex on the solid support member.
After washing the unreacted material from the support member, the binary complex is contacted with a second, labeled antibody that is also reactive with the selected antigen. If the binary complex is present on the solid support, this second antibody reacts with and becomes bound to the antigen component of the complex to form an antibody-antigen-antibody tertiary complex. After washing the solid support member to remove any of the second antibody that did not react with the selected antigen, the support member is tested for the second antibody by any of a variety of analytical techniques.
Diagnostic procedures of the above-outlined type have been successfully developed using polyclonal and monoclonal antibodies to detect antigens associated with particular diseases with a very high degree of reliability. Monoclonal antibodies are made via a process, referred to as hybridoma technology, in which hybridomas are formed by the fusion of short-lived antibody producing cells (usually spleen cells) and long-lived myeloma cells to produce long-lived antibody synthesizing cell lines. Each hybrid cell line produces a unique and characteristic antibody that has the ability to bind, with a very high degree of specificity, to a single type of antigen.
The cells that are fused to form a particular hybridoma cell line can be selected or treated so that the monoclonal antibody synthesized by that cell line will bind only to a chosen antigen. If such an antibody is used in the above-discussed procedure as either the first or second antibody, then the antibody-antigen-antibody tertiary complex will form on the solid support member, with a very high degree of accuracy, if and only if the chosen antigen is present in the sample liquid solution. Polyclonal antibodies with the appropriate affinity and specificity may also be used to detect antigens with a very high degree of accuracy.
Procedures of the above-outlined general type have been successfully employed on a commercial basis in the laboratory to test for human and animal diseases, among other things. Because of the relative simplicity and accuracy of procedures of this type, it would be very desirable to provide similar tests to diagnose plant diseases. Unique problems have been encountered, though, in developing commercially practical procedures, of the above-discussed general type, for the field diagnosis of plant diseases. For instance, it has been difficult to develop a simple and inexpensive yet field-effective technique for preparing a suitable liquid solution containing an effective quantity of a plant material that may be tested to indicate reliably whether the plant is infected with a particular antigen.